Molecular characterization and DNA methylation profile of Libyodrilus violaceous from oil polluted soil

Studies on earthworms using molecular markers are rare in Africa except a handful from South Africa. Reports on Libyodrilus violaceous, an earthworm found in West Africa are available including their metal tolerance and bioaccumulation capacity but their molecular characterization and ecotoxicology studies are scarce. In this study, triplicate L. violaceous specimens were collected from four locations within a petroleum polluted site and one in a control site, ≃1Km away from point of spill. DNA was extracted and 18S rRNA and 16S rRNA genes were amplified and sequenced. DNA methylation of their 18S rRNA gene was determined using Methylation specific PCR (MSP) method. Phylogenetic trees generated for 18S rRNA and 16S rRNA genes grouped L. violaceous within the Eudrilidae family concurrent with its conventional grouping and MSP results indicate no methylation in L. violaceous population from this site.


INTRODUCTION
Earthworms are ecologically important soil fauna hence their role as terrestrial bioindicators. The estimated earthworm species worldwide was approximated as 3000 in 2007 [1] and 3700 species in 2008 [2]. With molecular methods, there are estimated; 67 species in Greece [3], 90 species in Balkan Peninsula [4], 28 species in Guadeloupe [5] and 212 species in Vietnam [6]. Figures on indigenous African earthworm species are lacking and their sequences are few in repositories.
The conventional earthworms' taxonomy is labour-intensive, takes time and requires expertise. Some features of earthworms used for earthworm taxonomy at family level are now unreliable [1]. Molecular approach for characterization and earthworm ecological studies outweighs that of conventional methods [7][8][9]. Others argue that there are earthworm identification mismatch in both methods. Depending on one DNA region for molecular identification could be inaccurate as regions like mitochondria are known for their paraphyly and polyphyly [10][11][12]. More studies to investigate and corroborate previous classifications are therefore necessary.
DNA barcoding [13] using 18S rRNA and 16S rRNA genes and DNA methylation profiling (MSP) are the molecular methods used in classification and ecotoxicology in this study. 18S rRNA gene is recommended for taxonomic groupings of eukaryotes [14] while 16SrRNA gene are commonly used in classifying prokaryotes [15] and such sequences are found in the GenBank [16,17]. Methylation occurs in the DNA as organisms respond to environmental stress like heavy metal pollution [18,19] resulting in gene expressions or repressions [20]. Kille, showed DNA methylation among earthworm species in arsenic polluted site [21]. Gross also showed the variability of the 18S rRNA locus of three fish species seen in their chromosomal rearrangement [22] and Li established cysteine methylation of 18SrDNA gene in Arabidopsis from Nickel polluted soil [23].
The most common African earthworm family is Eudrilidae [24], endemic to tropical and sub-tropical soils, a taxa with 45 genera and 350 species [25] but only 34 nucleotide sequences and 11 protein sequences of the Eudrilidae family are found in NCBI /DDBJ/EMBL/GenBank. Beddard [26] first described Libyodrilus violaceous as a West African earthworm species isolated from Lagos, conventionally classified under the family Eudrilidae [24], subfamily Polytoreutinae and genus Libyodrilus. They have 'acorn-shaped' enlarged central chamber [27], ventrally located tubular structured prostrates and prostratic pores with male pore at the 17/18 intersegmental furrow. Their spermatheca also combines with ovarian duct as an organ at segment 14. Unlike other members of the family, they lack dorsal pores and nephridiopore [26]. Other genera in the family Eudrillidae include Hyperiodrilus and Polytoreutus.
L. violaceous are found in muddy soils abundantly during the wet seasons [28]. They appear purplish and luminescent occurring mostly twined together forming rope-like bundles [26,[29][30][31][32]. The species is found in Cameroon and distributed from the middle belt spanning down the southern part of Nigeria [32]. The importance of this earthworm species are highlighted in investigations including physiology [33], ecology [28], nutrition [34], ecotoxicology [35][36][37][38][39] especially its ability to tolerate polluted soils [28,38] and metal bioaccumulation potential [40]. Except from a few molecular reports on L. violaceous [38,40], there is dearth of information in its molecular characterization and ecotoxicology. This study involved molecular characterization of L. violaceous using 16SrRNA and 18SrRNA genes and determination of DNA methylation in L. violaceous specimens from a site eight years post impact of oil spills in a pipeline vandalized area in Lagos State, Nigeria [28]. Earthworms were collected by hand from 0 -15cm depth of soil surface and allowed to depurate for 24hrs in the laboratory. 3-5mm of the anterior end of each earthworm was cut and remaining gut content removed; this portion was preserved in RNA shield (ZYMO RESEARCH) for further molecular procedures. Earthworm specimens were identified conventionally by Prof.Owa, an earthworm taxonomist from the Zoology Department, Osun State University, Nigeria [28].

Molecular characterization Earthworm Collection
DNA Extraction, PCR amplification and Sequencing of 18S rRNA and 16S rRNA genes: ≤25mg anterior portion of each sample was cut and grinded in Liquid Nitrogen. DNA extraction was done using Quick-DNA TM Universal kit (ZYMO RESEARCH) following the manufacturer's procedure. Isolated DNA were amplified with final volume of 20 µl using 2 µl (10mM) of primers: 16SrRNA; F: CGA CTG TTT AAC AAA AAC AT (ewA) and R: CGC GGT CTG AAC TCA GCT CAT G (ewF) (≃ 520 bp) [41] and 18SrRNA; F: CAG CAG CCG CGG TAA TTC C (F-566), R: CCC GTG TTG AGT CAA ATT AAG C (R-1200) (≃650 bp) [42]. Also 2µl DNA template, 10µl PCR Master mix (Phusion High-Fidelity) and 4 µl nuclease H2O. The PCR conditions were as follows; one cycle of 10 mins at 95 o C 34 cycles of 30s at 95 o C 40s at 48 o C and 50 o C for 16S rRNA and 18S rRNA respectively, 1 min of 72 o C; then 5 mins of 72 o C. Gel electrophoresis using 1% (w/v) Agarose gel stained with ethidiumbromide (10mg/ml) was done to visualize the PCR amplicons. Representative PCR amplicons were sequenced using Sanger sequencer (Central analytical facility, Stellenbosch University).

Phylogenetic analyses:
The 18S rRNA and 16S rRNA nucleotide sequence reads were compared with reference sequences obtained from NCBI GenBank using the Basic Local Alignment Search Tool (BLAST). Sequence alignments were done with MAFFT program using the L-INS-i algorithm (maximum likelihood) (ultrafast bootstrap with 1000 replicates). The Phylogenetic trees were generated with the command-line version of the IQ-tree software version 1.5.5 [43][44][45][46].

RESULTS
16S rRNA and 18S rRNA genes were successfully amplified and viewed at the expected sizes (≃520bp and ≃650bp respectively) [41][42]. The partial nucleotide sequences of the two genes of the representative L.violaceous specimens are deposited in DDBJ/EMBL/GenBank with accession numbers KY114795.1 (18SrRNA) and KY114796.1 (16S rRNA). The 18S rRNA nucleotide sequence of L.violaceous shows this earthworm species had close genetic relationship with Eudriloides sp. Polytorentus finni and Hyperiodrilus sp. (Table 1). Similarly, the 16SrRNA nucleotide sequence BLAST indicates close identities with Eudriloides sp.; Hyperiodrilus sp; Metaphire sieboldi; Dichogaster sp and P. finni (Table 2). The molecular classification of L. violaceous with 18S rRNA gene is depicted in the phylogenetic tree below (Fig. 1). The tree has two major clusters, the first comprising of the Eudrilidae group with the species; L. violaceous, P. finni, Hyperiodrilus sp. and Eudriloides sp. and the second with species of the families Microchaetidae, Tritogeniidae, Hormogastridae, Glossoscolecidae, Megascolecidae, and Acanthodrilidae of the superfamily Megascolecoidae. The molecular classification of L.violaceous using16S rRNA gene is depicted in the phylogenetic tree below (Fig. 2). The tree shows two major sister clusters, the first, a clade consisting of Eudrilidae sp. and Eudrilus eugeniae with a common root. The second sister cluster has three sub-clusters with the first having Dichogaster sp. and Metaphire sieboldi nested in one clade and the second clade having L. violaceous, Hyperiodrilus sp. and P. finni. The third subcluster shows the remaining reference earthworm spp. There was no variation in the specimens of L. violaceous within the polluted site compared to the control.

DISCUSSION
The 16S rRNA and 18S rRNA genes were successfully amplified. The percentage coverage and identity of the sequence reads of both genes and phylogenetic trees corroborate the conventional classification of L. violaceous as a Eudrillidae.
The closest relative of L. violaceous is Eudriloides sp. It originates from Kenya, central Africa. James and Davidson [47] had reported Eudriloides sp. as more evolutionary related with Hyperiodrilous sp. in the 18S+28S partitioned analysis, Bayesian phylogram. They also reported this species to be equally related to P. finni and Hyperiodrilus africanus in a 28S gene tree. P. finni and Hyperiodrilus sp. are of the genus Polytoreutus and Hyperiodrilus, respectively, of the family Eudrilidae [24]; all four species are seen clustered in one clade in our study (Fig. 1) and is consistent with the conventional grouping of the species [26,29].
The second subcluster shows a clade of Glossoscolecidae; Fimoscolex sp., and Glossodrilus sp. [53,54]; The closeness of Glossoscolecidae to Eudrilidae had earlier been highlighted as they have similar ejaculatory structure [47,55] and they possess euprostate and paired dorsal calcium carbonate gland in either segment 12 or 13 [47]. The second subcluster also shows a clade of the family Megascolecidaeis; Amynthus sp, Metaphire exilis and Terrisswakerius sp. and another of the Acanthodrilidae family; Dichogaster saliens and Dichogaster sp. All species in the second subcluster are from the super family Megascolecidae. The phylogenetic tree generated clusters that indicate a common origin of all species being oligochaetes; of the polyphyletic order Crassiclitellata [56][57].
The phylogenetic tree for the 16S rRNA gene ( fig. 2) shows weak clustering of Eudrilidae sp. and Eudrilus eugeniae species separate from the other clade of L. violaceous, Hyperiodrilus sp. and P. finni although they are all of the Eudrilidae family. This could be due to the polyphyletic nature of the Eudrilidae family [47,51], it could also be because 16S rRNA gene is best for prokaryotic classification [15]. Dichogaster sp. and Metaphire sieboldi in the second cluster are conventionally of the super family Megascolecidae. The third sub-clade clusters Aporrectodea georgii, Eisenia fetida, Bimastos zeteki, Eiseniella tetraedra, Lumbricus rubellus and Lumbricus terristris (of the family Lumbricidae) with Hormogaster praetiosa (of the same super family Lumbricoidea).
The phylogenetic grouping in this study (Fig. 1, 2) supports the conventional classification of L. violaceous with the18S rRNA gene as more reliable than the 16S rRNA gene for its molecular taxonomic grouping. 18S rRNA gene is acclaimed as more appropriate for eukaryotic classification than 16S rRNA gene [14]. Among the reference earthworm species, the closest evolutionary relation of L. violaceous for 18S rRNA partial nucleotide sequence was Eudrilidae sp, although the taxonomy to the specie level of Eudrilidae sp. still remains imprecise. Chang [58] had alleged many incorrect earthworm species sequences in the GenBank which results in misleading conclusions. This however, cannot be adjudged so in our conclusion since the phylograms for L. violaceous 18S rRNA and 16S rRNA indicate evolutionary differences between L. violaceous and Eudrilidae sp.
Although pollutants like metals are known to modify DNA [59] or cause epigenetic responses like methylation and locus variability [21][22][23], there were no methylations in the specimens sampled in this study. The sampling site is an oil polluted site post incident of oil spill and inferno earlier reported to have heavy metal concentrations [60], it possibly had undergone natural attenuation over the 8yrs [61,62]. From the earlier report, levels of Zn, Cd, Mn, Ni, V, Pb, Cu and Cr in both polluted and control sites were all below the FEPA standard threshold value [62] hence could be why the metals did not cause epigenetic impact on the 18S rRNA gene of this earthworm species. It could also be due to the fact that the metals monitored do not cause epigenetic impact on earthworm species since none of these metals have been reported to cause methylation of 18SrRNA gene in any available literature. Arsenic is one of the few metals reported to cause epigenetic responses resulting in variation among species of organisms [21].